Primate embryonic stem (ES) cells and the recently-described induced pluripotent stem cells (iPS) (collectively, “pluripotent cells”) can proliferate without limit and can differentiate into each of the three embryonic germ layers. ES cells are stem cells found in or derived from embryos. ES cells are capable of differentiation into most, if not all, of the differentiated cell types of a mature body. It is understood that iPS cells behave in culture essentially the same as ES cells. iPS cells and ES cells express one or more pluripotent cell-specific marker, such as Oct-4, SSEA-3, SSEA-4, Tra 1-60, Tra 1-81, and Nanog (Yu et al. Science, Vol. 318. no. 5858, pp. 1917-1920 (2007), incorporated by reference here in its entirety) Also, recent findings of Chan, suggest that expression of Tra 1-60, DNMT3B, and REX1 can be used to positively identify fully reprogrammed iPS cells, whereas alkaline phosphatase, SSEA-4, GDF3, hTERT, and NANOG are insufficient as markers of fully reprogrammed iPS cells. (Chan et al., Nat. Biotech. 27:1033-1037 (2009), incorporated by reference here in its entirety). Subsequent references herein to primate or human ES cells and the like are intended to apply with equal force to iPS cells.
Pluripotent cells are of high interest to the research community and regenerative industry because they are capable of indefinite proliferation in culture. Thus, at least in principle, pluripotent cells are capable of supplying cells and tissues for replacement of failing or defective human tissue. This is why the existence of human pluripotent stem cells in culture offers the potential of unlimited amounts of human cells and tissues for use in a variety of therapeutic protocols to assist in human health. It is envisioned in the future that human pluripotent stem cells will be proliferated and directed to differentiate into specific lineages so as to develop differentiated cells or tissues which can be transplanted into human bodies for therapeutic purposes. Human pluripotent stem cells and the differentiated cells that may be derived from them are also powerful scientific tools for the study of human cellular and developmental systems.
Specifically, the basic techniques to create and culture human ES cells have been described. The previously reported techniques do work, but there are limitations and drawbacks to many of the procedures currently used to culture human ES cells. One limitation of particular concern is that most existing human ES cell lines have been, to one degree or another, exposed directly to mouse cells or to a medium in which mouse cells have been previously cultured. This is because the original techniques for generating and culturing human ES cells required using mouse embryonic fibroblast (MEF) feeder cells as a feeder layer on which human ES cells could be cultured. The fibroblast feeder cells act, through some as yet incompletely understood mechanism, to encourage the stem cells to remain in an undifferentiated state. A by-product of such techniques is that some human ES cells from existing cell lines were found to exhibit the sialic residue Neu5Gc, which is not normally made by human cells but is made by murine cells, and has received much attention in the press.
Later, it was discovered that the benefits MEF feeder cells provide to stem cells grown in culture could be obtained by exposing stem cells to “conditioned medium.” Conditioned medium is a stem cell culture medium in which feeder cells, such as MEFs, had been previously cultured. In conditioned medium, either the feeder cells impart some factor to the medium or remove some factor from the medium, which allows stem cells to be cultured in an undifferentiated state. Although the mechanisms involved in the beneficial effects of conditioned medium have not been fully elucidated, the result is that conditioned medium can be used to maintain stem cells in culture with minimal differentiation. Both direct growth of human pluripotent stem cells on murine feeder cells and the use of conditioned media raise the concern that one or more agents, such as a virus, could be transmitted from the mouse cells to the human pluripotent stem cells. If one of the applied objectives of human pluripotent stem cell cultures is to create cells and tissues which can ultimately be transplanted into a human body, it is highly desirable to generate stem cells that have never been exposed to cells of another species or to media which have been used to culture cells of another species. Accordingly, defining a culture condition, which will permit the proliferation and culture of human pluripotent stem cells without a fibroblast feeder layer, is of great interest in the continued development of techniques for the long term culture of human pluripotent stem cells.
Several medium formulations will permit human pluripotent stem cells to remain undifferentiated for some time, but the undifferentiated state often fails to maintain itself. The inventors here have found several medium formulations that permit the cultivation of human pluripotent stem cells for one or two passages without severe differentiation, but then the cells differentiate rapidly upon subsequent passages. In particular, “passage” is defined herein as the growth of human pluripotent stem cells from an initial seed culture in a culture vessel to confluence in the same culture vessel. The inventors have come to believe that in order for a culture medium to truly support the indefinite proliferation of human pluripotent stem cells without differentiation and without fibroblast feeder cells or medium that is conditioned therewith, the medium must be capable of supporting culture of human pluripotent stem cells in a substantially uniform and undifferentiated state for at least five passages. It is also important that the cultures remain relatively homogenous and undifferentiated throughout the culture period and retain all of the important characteristics of human pluripotent stem cells.
A characteristic trait of human pluripotent stem cells in culture is that, if conditions are less than ideal, the cells have a tendency to differentiate. It is easy to induce human pluripotent stem cells to differentiate, while it is challenging to maintain human pluripotent stem cells in an undifferentiated state in culture. Most culture conditions will result in some level of unwanted differentiation, particularly around the periphery of the growing pluripotent stem cell colony. While pluripotent stem cells can be cultured with some degree of unwanted differentiation, the objective is to define a culture condition that permits the culture to remain as undifferentiated as possible, i.e., with as few differentiated cells as possible. To achieve this objective, the inventors have used particularly stringent standards to define conditions that will support the indefinite culture of undifferentiated pluripotent stem cell cultures, as described below.
The state of differentiation of a stem cell culture can be assessed by morphological characteristics. Undifferentiated stem cells have a characteristic morphology, i.e., small and compact cells with clearly defined cell borders, a morphology which can be easily seen by examination of a stem cell culture under a microscope. By contrast, cells which have differentiated appear larger and more diffuse with indistinct cell borders. While some differentiated cells can, and normally do, appear at the margin of colonies of undifferentiated cells, the optimal stem cell culture is one that proliferates in the culture vessel with only minimal numbers of cells at the periphery of the culture appearing to be differentiated. With experience, stem cell researchers can judge the status of differentiation and health of human pluripotent stem cell cultures visually with good accuracy.
In addition to morphological characteristics, biochemical cell markers are routinely used to track the status of ES cells as undifferentiated. For example, transcription factor Oct-4 is regarded as the most reliable marker of undifferentiated status for ES cells. Oct-4 is also one of the first markers lost as undifferentiated cells begin to differentiate. Other biochemical markers used to track the status of undifferentiated primate ES cells and human ES cells include: SSEA-3, SSEA-4, Tra 1-60, and Tra 1-81 and alkaline phosphatase, but not SSEA-1. In contrast, undifferentiated mouse ES cells express SSEA-1, but not SSEA-3, SSEA-4, Tra 1-60, and Tra 1-81. Tra 1-60, and Tra 1-81 are antibodies to extracellular matrix molecules synthesized by undifferentiated pluripotent stem cells, which are also markers for human ES cells. Further, it has been shown that Nanog is a transcription factor that is essential for the maintenance of pluripotency and self renewal in pluripotent stem cells.
In addition to stringent standards required for conditions that will support the indefinite culture of undifferentiated pluripotent stem cell cultures, the conditions needed for derivation of new lines of human pluripotent stem cells is an even more stringent. This is because some culture conditions which support the expansion and growth of existing stem cells lines have not proven sufficient for use in the derivation of new human pluripotent stem cell lines. It appears that the capacity to support the initiation of new lines of stem cells is a capacity that not all stem cell culture conditions feature. Accordingly, media to support initiation, proliferation and culture of undifferentiated primate stem cells are desired by the stem cell industry.